The present invention is directed to improved adenoviral helper vectors that facilitate the production of pseudoadenoviral vectors (PAV), wherein the helper vectors themselves cannot be packaged into viral particles efficiently. While the helper vectors of the present invention are engineered, such that during PAV manufacture they are packaging defective, they may provide replication functions and viral structural proteins in trans for PAV. The helper vectors of the present invention comprise recombinase protein recognition sequences, wherein said recognition sequences are inserted into regions of the helper vector genome to allow for separation of the replication and packaging elements of the helper vector. Action by the cognate recombinase on such recombinase protein recognition sequences create an inversion or deletion of the genome upon recombination, thereby positioning the packaging elements such that the helper genome cannot be packaged. The invention is also directed to improved cell lines for the production of PAV which facilitate PAV stock production. The improved producer cell lines are stably transfected with a novel PAV. The combination of the novel helper vectors of the present invention, the novel PAVs and the improved cell lines facilitate PAV stock production and make the invention adaptable to large scale commercial production of PAV stocks.
Adenoviral vectors for use to deliver transgenes to cells for applications such as in vivo gene therapy and in vitro study and/or production of the products of transgenes, commonly are derived from adenoviruses by deletion of the early region 1 (E1) genes (Berkner, K. L., Curr. Top. Micro. Immunol. 158:39-66, 1992). Deletion of E1 genes renders such adenoviral vectors replication defective and significantly reduces expression of the remaining viral genes present within the vector. However, it is believed that the presence of the remaining viral genes in adenoviral vectors can be deleterious to the transfected cell for one or more of the following reasons: (1) stimulation of a cellular immune response directed against expressed viral proteins, (2) cytotoxicity of expressed viral proteins, and (3) replication of the vector genome leading to cell death.
One solution to this problem has been the creation of pseudoadenoviral vectors (PAVs), which are adenoviral vectors derived from the genome of an adenovirus that contain minimal cis-acting nucleotide sequences required for the replication and packaging of the vector genome and which can contain one or more transgenes (See, U.S. Pat. No. 5,882,877 which covers pseudoadenoviral vectors (PAV) and methods for producing PAV, incorporated herein by reference). Such PAVs, which can accommodate up to about 36 kb of foreign nucleic acid, are advantageous because the carrying capacity of the vector is optimized, while the potential for host immune responses to the vector or the generation of replication-competent viruses is reduced. PAV vectors contain the 5xe2x80x2 inverted terminal repeat (ITR) and the 3xe2x80x2 ITR nucleotide sequences that contain the origin of replication, and the cis-acting nucleotide sequence required for packaging of the PAV genome, and can accommodate one or more transgenes with appropriate regulatory elements, e.g. promoters, enhancers, etc.
Adenoviral vectors, such as PAVs, have been designed to take advantage of the desirable features of adenovirus which render it a suitable vehicle for delivery of nucleic acids to recipient cells. Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb, which has been well-characterized through studies in classical genetics and molecular biology (Hurwitz, M. S., Adenoviruses Virology, 3rd edition, Fields et al., eds., Raven Press, New York, 1996; Hitt, M. M. et al., Adenovirus Vectors, The Development of Human Gene Therapy, Friedman, T. ed., Cold Spring Harbor Laboratory Press, New York, 1999). The viral genes are classified into early (designated E1-E4) and late (designated L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins. The demarcation of these events is viral DNA replication. The human adenoviruses are divided into numerous serotypes (approximately 47, numbered accordingly and classified into 6 groups: A, B, C, D, E and F), based upon properties including hemaglutination of red blood cells, oncogenicity, DNA and protein amino acid compositions and homologies, and antigenic relationships.
Recombinant adenoviral vectors have several advantages for use as gene delivery vehicles, including tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (Berkner, K. L., Curr. Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapy 1:51-64, 1994).
PAVs have been designed to take advantage of the desirable features of adenovirus which render it a suitable vehicle for gene delivery. While adenoviral vectors can generally carry inserts of up to 8 kb in size by the deletion of regions which are dispensable for viral growth, maximal carrying capacity can be achieved with the use of adenoviral vectors containing deletions of most viral coding sequences, including PAVs. See U.S. Pat. No. 5,882,877 of Gregory et al.; Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Parks et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996; Lieber et al., J. Virol. 70:8944-8960, 1996; Fisher et al., Virology 217:11-22, 1996; U.S. Pat. No. 5,670,488; PCT Publication No. WO 96/33280, published Oct. 24, 1996; PCT Publication No. WO 96/40955, published Dec. 19, 1996; PCT Publication No. WO 97/25446, published Jul. 19, 1997; PCT Publication No. WO 95/29993, published Nov. 9, 1995; PCT Publication No. WO 97/00326, published Jan. 3, 1997; Morral et al., Hum. Gene Ther. 10:2709-2716, 1998.
Since PAVs are deleted for most of the adenovirus genome, production of PAVs requires the furnishing of adenovirus proteins in trans which facilitate the replication and packaging of a PAV genome into viral vector particles. Most commonly, such proteins are provided by infecting a producer cell with a helper adenovirus containing the genes encoding such proteins. However, such helper viruses are potential sources of contamination of a PAV stock during purification and can pose potential problems when administering the PAV to an individual if the contaminating helper adenovirus can replicate and be packaged into viral particles.
It is advantageous to increase the purity of a PAV stock by reducing or eliminating any production of helper vectors which can contaminate preparation. Several strategies to reduce the production of helper vectors in the preparation of a PAV stock are disclosed in U.S. Pat. No. 5,882,877, issued Mar. 16, 1999; U.S. Pat. No. 5,670,488, issued Sep. 23, 1997 and International Patent Application No. PCT/US99/03483, incorporated herein by reference. For example, the helper vector may contain mutations in the packaging sequence of its genome to prevent its packaging, an oversized adenoviral genome which cannot be packaged due to size constraints of the virion, or a packaging signal region with binding sequences that prevent access by packaging proteins to this signal which thereby prevents production of the helper virus.
Other strategies include the design of a helper virus with a packaging signal flanked by the excision target site of a recombinase, such as the Cre-Lox system (Parks et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996; Hardy et al.,J. Virol. 71:1842-1849, 1997, incorporated herein by reference). Such helper vectors reduce the yield of wild-type levels.
Another hurdle for PAV manufacturing, aside from the problems with obtaining helper vector-free stocks, is that the production process is initiated by DNA transfections of the PAV genome and the helper genome into a suitable cell line, e.g., 293 cells. After cytopathic effects are observed in the culture indicating a successful infection, which may take up to from 2 to 6 days, the culture is harvested and is passaged onto a new culture of cells. This process is repeated for several additional passages, up to 7 times more, to obtain a modest cell lysate containing the PAV vector and any contaminating helper vector. See Parks et al., 1996, Proc. Natl. Acad. Sci. USA 93:13565-13570; Kochanek et al., 1996, Proc. Natl. Acad. Sci. USA 93:5731-5736. This lengthy process is not optimal for commercial scale manufacturing. Additionally, this process facilitates recombination and rearrangement events resulting in the propagation of PAV genomes with unwanted alterations.
Therefore, there is a need for improved helper vectors which promote the production of substantially helper vector-free PAV stocks and there is a need for improved cell lines which simplify the PAV stock production process while being adaptable to large scale commercial production.
In general, it is the object of the present invention to provide improved helper vectors and cell lines for the production of pseudoadenoviral (PAV) and other adenoviral vectors containing substantially reduced levels of contaminating helper vector. The invention provides for helper vectors for the production of substantially helper vector-free PAV stocks comprising recombinase recognition sequences (e.g. Cre/Lox, Flp/FRT system, and phage [xcfx86] C31) which, depending upon their arrangement within the helper vector, can prevent helper vector packaging. In a particularly preferred embodiment of the invention, the recombinase recognition sequences employed are those from phage C31. Phage C31 recombinase appears to be able to promote recombination/excision in a manner similar to Cre/Lox or Flp, but will not perform the reverse reaction to re-insert the excised piece. In theory, this recombinase should eliminate the generation of mutations and re-insertions that make the helper resistant to recombinase activity.
The invention also provides for improved cell lines for the production of substantially helper vector-free PAV stocks comprising a stably introduced novel circular PAV genome into the cell.
The term xe2x80x9cPAVxe2x80x9d is used generally to refer to recombinant adenoviral vectors from which a significant portion of the adenoviral genome has been deleted. In addition to fully deleted adenoviral vectors, such as those described in WO94/12649 [Gregory et al.]; WO99/64577 [Morsy et al.]; WO96/33280 [Zhang et al.]; WO 98/54345 [Zhang et al.]; WO95/29993 [Nabel et al.]; Balague et al., Blood, 95:820-828 (2000); and Fisher et al., Virology, 217:11-22 (1996); Vincent et al., Nature Genetics, 5:130-134 (1993); Sandig et al., PNAS USA 97:1002-1007(2000); Morsey et al., Molecular Medicine Today: Reviews, 1999:18-24 (1999), other partially deleted adenoviral vectors may be used in the methods of the present invention in place of PAVs. These include those described in U.S. Pat. No. 6,063,622 [Chamberlain et al.], U.S. Pat. No. 6,093,567 [Graham et al.], U.S. Pat. No. 6,093,567 [Gregory et al.], WO99/57296 [Wadsworth et al.] and WO00/12740 [Amalfitano et al.], the disclosures of all of these publications are hereby incorporated herein by reference.
During the production of recombinant adenoviral stock, the functions of the deleted portion of the adenoviral genome may be supplied in trans, usually via co-infection with a replication incompetent helper virus, or by culturing the virus in a stock of transfected packaging cells. The methods and materials of the present invention are useful for methods of producing recombinant adenoviral stocks with improved efficiency and safety characteristics.
In one embodiment, the helper vector provides adenovirus (Ad) functions in trans and comprises an adenoviral genome containing recombinase recognition sequences placed in an inverted orientation relative to each other such that the action of the recombinase on the cognate recombinase recognition sequences inverts the central portion of the helper vector genome and displaces the packaging elements from their close proximity to both or either of the 5xe2x80x2 and 3xe2x80x2 ITR. This displacement results in the packaging elements being moved to a distance of approximately 3,000 nucleotides from the ITR. Since the distance from the ITR required for the proper functioning of the adenovirus (e.g. Ad5) packaging motif is approximately 600 nucleotides from the ITR, such a recombination event compromises packaging of the helper vector while leaving intact the genes encoding replication functions and structural proteins necessary for the packaging of the PAV.
In another embodiment, the recombinase recognition sequences in the helper vector are placed in the same orientation and at opposite ends of the helper genome. Action of the recombinase on the cognate recombinase recognition sequences leads to formation of a circular helper genome molecule and a separate short linear DNA that contains the left and right ITRs, i.e. the ITRs are excised entirely out of the genome. The excision of the ITRs results in a helper genome which cannot be packaged because the packaging sequences are no longer adjacent to an ITR as required. Alternative replication origins, such as those from SV40 and EBV, may be incorporated into the helper genome to allow for continued replication of the circular helper genome. Additionally, the requisite nucleotides encoding required replication proteins to drive replication of the helper genome, such as SV40 large T antigen or EBNA which bind to the origin and initiate replication, may also be provided.
In a further embodiment, the helper vector comprises a packaging signal region flanked by recombinase (e.g. FRT, or phage [xcfx86] C31 target sequences) such that binding of a recombinase at the recombinase nucleotide binding sequences results in excision of the packaging signal region from the helper vector. The genome of the helper vector may further comprise stuffer sequences which may improve excision of the packaging elements therefrom.
Another object of the invention is to provide improved cell lines for the production of PAV stocks. Such improved cell lines comprise a circular PAV genome comprising a bacterial plasmid genome (e.g. pBR322), or an origin of replication and drug resistance sequences derived from a bacterial plasmid genome (e.g. pBR322) to allow propagation of the PAV in bacteria, and eukaryotic and/or viral replication origin sequences (e.g. EBV sequences) to allow replication of the PAV genome within the mammalian producer cell line. PAV production is initiated by infection of the cell line with a suitable helper vector, such as those described above comprising recombination recognition sequences for the prevention of helper vector production. The helper vector provides, in trans, replication and packaging functions required for the production of PAV stocks. Since the circular PAV genome has its ITR sequences embedded (i.e. not at a terminus), its replication and packaging efficiencies are low. However, it is known that at a low frequency, excision of at least one ITR occurs which allows for efficient replication and packaging at its occurrence. Graham, 1984, EMBO J. 3:2917-2922; Matani et al, 1995, Proc. Natl. Acad. Sci. USA 92:3854-3858; Hardy et al., 1997, J. Virol. 71:1842-1849; Fisher et al., 1996, Virol. 217:11-22). Alternatively, the circular PAV genome is constructed such that there is a head-to-tail duplication of the ITR adjacent to the packaging sequence as would exist when Ad DNA is being replicated as a linear concatenate. See, e.g., Graham, 1984, EMBO J. 3:2917-2922; Fisher et al., 1996, Virol. 217:11-22. Excision of the ITR from this configuration is more efficient than from a single ITR. The circular PAV genome may comprise restriction endonuclease sites at positions just beyond the ITRs which, when excised by a restriction endonuclease, make the ITRs accessible and allow for efficient replication and packaging of the PAV. The helper vector may provide nucleotide sequences encoding for the appropriate restriction endonuclease (e.g. RsrII, I-Ceu I, I-Ppo I; available from New England Biolabs, Beverly, Mass.).